The present invention provides oligonucleotides labelled with substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY.RTM. fluorophore) compounds for performing the Taqman assay. The ability to detect DNA and specific sequences of DNA is critical for understanding the function and control of genes and for diagnosis of genetically-inherited diseases. Native DNA consists of two linear polymers or strands of nucleotides. Each strand of DNA is a chain of nucleotides linked by phosphodiester bonds. The two strands are held together in an antiparallel orientation by hydrogen bonds between complementary bases of the nucleotides of the two strands: deoxyadenosine (A) pairs with thymidine (T) and deoxyguanosine (G) pairs with deoxycytidine (C).
A significant advance in DNA manipulation was the development of the polymerase chain reaction (PCR) technique as disclosed in U.S. Pat. Nos. 4,683,195; 4,683,195; and 4,800,159. The term "polymerase chain reaction" or "PCR" refers generally to the procedure involving: (1) treating extracted DNA to form single-stranded complementary strands; (2) adding a pair of oligonucleotide primers, wherein one primer of the pair is substantially complementary to part of the sequence in the sense strand and the other primer of each pair is substantially complementary to a different part of the same sequence in the complementary antisense strand; (3) annealing the paired primers to the complementary sequence; (4) simultaneously extending the annealed primers from a 3' terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer wherein said extension products after separation from the complement serve as templates for the synthesis of an extension product for the other primer of each pair; (5) separating said extension products from said templates to produce single-stranded molecules; and (6) amplifying said single-stranded molecules by repeating at least once said annealing, extending and separating steps.
Detection methods generally employed in standard PCR techniques use a labeled probe with the amplified DNA in a hybridization assay. Other assays include the use of fragment length polymorphism (PCR FLP), hybridization to allele-specific oligonucleotide (ASO) probes (Saiki et al., Nature 324:163 (1986)), or direct sequencing via the dideoxy method (using amplified DNA rather than cloned DNA). The standard PCR technique operates by replicating a DNA sequence positioned between two primers, providing as the major product of the reaction a DNA sequence of discrete length terminating with the primer at the 5' end of each strand. Thus, insertions and deletions between the primers result in product sequence of different lengths, which can be detected by sizing the product in PCR-FLP. In an example of ASO hybridization, the amplified DNA is fixed to a nylon filter (by, for example UV irradiation) in a series of "dot blots", then allowed to hybridize with an oligonucleotide probe labeled with HRP under stringent conditions. After washing, tetrametholbenzidine (TMB) and hydrogen peroxide are added: HRP oxidizes the hydrogen peroxide, which in turn oxidizes the TMB to a blue precipitate, indicating hybridized probe.
While the PCR technique as presently practiced is an extremely powerful method for amplifying nucleic acid sequences, the detection of the amplified material requires additional manipulation and subsequent handling of the PCR products to determine whether the target DNA is present. It is desirable to decrease the number of subsequent handling steps required currently for the detection of amplified material.
Holland, et al., PNAS 88:7276-7280 (1991) describe an assay known as a Taqman assay. The 5'.fwdarw.3' exonuclease activity of Taq polymerase is employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of the probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'.fwdarw.3' exonuclease activity of Taq polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods. A version of this assay is also described in Gelfand et al., in U.S. Pat. No. 5,210,015. U.S. Pat. No. 5,210,015 to Gelfand, et al., and Holland, et al., PNAS 88:7276-7280 (1991) are hereby incorporated by reference.
Further, U.S. Pat. No. 5,491,063 to Fisher, et al., provides a Taqman-type assay. The method of Fisher et al. provides a reaction that results in the cleavage of single-stranded oligonucleotide probes labeled with a light-emitting label wherein the reaction is carried out in the presence of a DNA binding compound that interacts with the label to modify the light emission of the label. The method utilizes the change in light emission of the labeled probe that results from degradation of the probe. The methods are applicable in general to assays that utilize a reaction that results in cleavage of oligonucleotide probes, and in particular, to homogeneous amplification/detection assays where hybridized probe is cleaved concomitant with primer extension. A homogeneous amplification/detection assay is provided which allows the simultaneous detection of the accumulation of amplified target and the sequence-specific detection of the target sequence. U.S. Pat. No. 5,491,063 to Fisher, et al. is hereby incorporated by reference.
Lee, et al., NAR 21:3761-3766 (1993) describe nick translation PCR using fluorogenic probes. In this assay, two probes were used to detect mutant and wildtype cystic fibrosis alleles. Lee, et al., NAR 21:3761-3766 (1993) is incorporated herein by reference.
The present invention is drawn to provides oligonucleotides labelled with substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY.RTM. fluorophore) compounds for performing the Taqman assay.